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Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse D_H-Less Heavy Chain Gene Usage and Avidity Maturation^1,2

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India

Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA^3–286) from strain R36A. Abs to PspA^3–286 were encoded by diverse V_H and V families/genes. The H chain CDR3 and L chain CDR3 lengths were 3–13 (7.8 ± 0.5) and 8–9 (8.7 ± 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D_H gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D_H gene-derived amino acids did not prevent anti-PspA^3–286 mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA^3–286 polyclonal Ab response in PspA^3–286- and R36A-immunized mice. Compared with PspA^3–286-immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D_H genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D_H-less Abs.

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¹ This work was supported by a grant from the Department of Biotechnology, Government of India (to D.S.) and a Senior Research Fellowship from the Council of Scientific and Industrial Research, India (to S.R.).

² The sequences presented in this article have been submitted to GenBank under accession numbers EU915613-EU915684.

³ Current address: Graduate School of Medicine, Kyoto University, Kyoto, Japan.

⁴ Address correspondence and reprint requests to Dr. Devinder Sehgal, Molecular Immunology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India. E-mail address: devinder{at}nii.res.in

⁵ Abbreviations used in this paper: PspA, pneumococcal surface protein A; AI, additivity index; FP, founder progenitor; FR, framework region; HCDR, H chain CDR; HP, hypothetical common precursor; LCDR, L chain CDR; N, nontemplate encoded; P, palindromic; RF, reading frame.

⁶ The online version of this article contains supplemental material.